1. The Field of the Invention
The present invention relates generally to analytical separation techniques such as field-flow fractionation. More specifically, the present invention relates to a method and a device for introducing samples in analytical separation apparatuses, and in particular to field-flow fractionation systems.
2. Related Application
This is a division of application Ser. No. 09/032,188, filed Feb. 27, 1998, on behalf of Yong Jiang, Ph.D., Marcia Elaine Hansen, Ph.D., Michael Eliot Miller, Ph.D., and Andreas Martin Kummerow, entitled Sample Focusing Device and Method, which is hereby incorporated herein by reference.
3. Present State of the Art
Field-flow fractionation is a separation and characterization technique that relies on the effects of an applied field on a sample that is carried by a fluid flow. This fluid flow moves down a channel that will hereinafter be referred to as the "channel". The stream flowing along the channel will be referred to by the term "channel flow".
The character and strength of the interaction between the species in the sample 15 and the field plays a decisive role in the separation. Species that more weakly interact with the field are more rapidly carried away by the fluid flow that moves perpendicular to the applied field. This leads to different retention times for different species in the sample. Field-flow fractionation was disclosed in U.S. Pat. No. 3,449,938, and it is an excellent technique to separate and characterize a great variety of species. Field-flow fractionation is also known as single phase chromatography, polarization chromatography and capillary hydrodynamic fractionation. These species include cells, subcellular particles, viruses, liposomes, protein aggregates, fly ash, colloids, industrial lattices and pigments, polymers, humic materials, proteins, and nucleic acid molecules such as DNA. Some of these species are dissolved in the fluid flow that carries the sample, whereas other species are better characterized as being suspended in the fluid flow. Consequently, the terms "carrier fluid" and "carrier" will hereinafter refer to the fluid flow that transports the sample species, regardless of the form in which such species are contained in the fluid medium (i.e., whether dissolved, dispersed, suspended or in any other form of aggregation in the fluid flow). Furthermore, terms such as "sample species", "particles" or "particle", and "component" or "components", will hereinafter characterize the entity or entities in the sample to be analyzed, or more particularly, in the sample that contains the entities to be separated. More particularly, these terms used in the specific context of field-flow fractionation refer to any sample species that can be retained and separated by any field-flow fractionation method, including rigid and deformable particles ranging in size from submicron to hundreds of microns, polymer molecules, aggregates and clusters, biological macromolecules, and particles including cells, DNA, proteins and any other molecules that are capable of analysis by field-flow fractionation. Consequently, these terms refer to the entities or components in the sample that is to be analyzed or separated, regardless of the nature, mass, size or any other specific characteristic of these entities, and the sample to be analyzed or separated is hereinafter referred to as the "sample".
The great variety of sample species that can be separated and characterized by field-flow fractionation makes this technique an important tool for solving problems in a plurality of fundamental and applied research areas that include biology, medicine, and material and environmental sciences. More specifically, field-flow fractionation has been applied to sample species whose masses span a 10.sup.15 -fold range. These species encompass molecules with a mass of about 600 Dalton and increasingly bigger entities up to particles of about 100 micrometers in diameter.
The choice of the applied field in field-flow fractionation depends on the particular property that controls the retention time of the sample species that is to be separated. The types of applied fields that can be used in implementing field-flow fractionation include thermal, gravitational, electric, and magnetic gradients. In addition, a cross flow with respect to the carrier is also used in flow field-flow fractionation, a very versatile and effective implementation of the field-flow fractionation principles. Other types of applied fields that have in fact been applied or that are of potential practical relevance as a driving force in field-flow fractionation include forces due to dielectrical, concentration gradient, photophoretic and shear effects. A short-hand notation that consists of the acronym FFF preceded by the name of the applied field is used hereinafter. Available commercial types of field-flow fractionation include flow FFF, thermal FFF, and sedimentation FFF. These types differ by the type of applied field. In flow FFF, the field that drives separation is a flow stream directed perpendicular to the channel flow longitudinal axis. A method and apparatus for flow FFF is described in U.S. Pat. No. 4,147,621. In thermal FFF, a thermal gradient is used as the field to drive separation. Acceleration is used to drive separation in sedimentation FFF. In particular, this acceleration is that of a centrifugal field in sedimentation FFF, and it is the gravitational field in gravitational FFF. Unless otherwise specified, the terms "field" or "applied field" will hereinafter refer to any applied field, to a cross flow, and to any appropriately generated potential gradient that creates a driving force that directs the sample species into a wall of the channel called the accumulation wall. Furthermore, the examples and illustrations offered herein refer in particular to flow FFF because this field-flow fractionation technique is currently established as a very versatile and effective technique. In addition, flow FFF has been characterized as the most universal of the field-flow fractionation methods. J. Calvin Giddings, Field-Flow Fractionation, Chemical and Engineering News, Vol. 66 (1988). pp.34-45; Particle Size Distribution II, ACS Symposium Series No. 472, S. Kim Ratanathanawongs, Inho Lee, and J. Calvin Giddings, Separation and Characterization of 0.01-50-.mu.m Particles Using Flow Field-Flow Fractionation, 1991, chapter 15, pp. 229-46.
For each applied field there are in turn a variety of operating modes. Each operating mode depends on the sample species separation mechanism. For example, sample species under the influence of an applied field may be subject to a diffusive, steric or hydrodynamic lift effects. Depending on which one of these effects is predominant, the field-flow fractionation operating mode is, respectively, a Brownian, steric or hyperlayer mode. Consequently, each appropriate choice of applied field and operating mode leads to a different field-flow fractionation subtechnique.
Whereas sample species separation according to mass or size is often the goal of field-flow fractionation, this is not the only possible application of field-flow fractionation. With the appropriate choice of applied field, a field-flow fractionation apparatus can perform as a microbalance sensitive to forces of 10.sup.16 N. Furthermore, field-flow fractionation permits the measurement of both particle size and density, from which a molar mass can be calculated. Other properties that can be calculated include particle diameter and charge. The high sensitivity of sedimentation FFF to very small amounts of adsorbed material permits the measurement of the mass and thickness of adsorbed layers. When the sample species population is heterogeneous in any of these properties, the different components are separated by field-flow fractionation on the basis of the heterogeneous property, and a distribution curve relative to this property is obtained. These and other background materials pertaining to field-flow fractionation have been described by Ronald Beckeff, John Ho, Yong Jiang, and J. Calvin Giddins, Measurement of Mass and Thickness of Adsorbed Films on Colloidal Particles by Sedimentation Field-Flow Fractionation, Langmuir, Vol. 7 (1991), pp. 2040-47; J. Calvin Giddings, Field-Flow Fractionation, Chemical and Engineering News, Vol. 66 (1988), pp. 34-45.
In a field-flow fractionation apparatus the carrier flows tinder laminar regime conditions along a narrow channel and a field is applied orthogonally to the carrier flow. One of the characteristics of a laminar flow is that the flow velocity profile is parabolic. Accordingly, the carrier moves slower near the walls and increasingly faster in regions closer to the channel center line of the channel along the longitudinal axis. As applied, the field drives sample species to different cross-sectional regions of the carrier flow, where they are transported with different momenta depending on the carrier flow region to which they are driven. The sample species are initially and ideally concentrated in a very small spot on one of the channel walls called the accumulation wall. In the course of flow displacement, particles that weakly interact with the field will move farther from the accumulation wall than the particles that strongly interact with the field, thus reaching sooner the regions of the carrier flow that move faster. These particles are carried downstream more rapidly than the particles that interact more strongly with the field. Therefore, rapidly swept particles are part of an outflow fraction that leaves the field-flow fractionation apparatus sooner than the fractions that contain the particles that more strongly interact with the field. More succinctly, the retention time of a particle depends on the interaction between the relevant property of the particle and the applied field. In the initial operation of the FFF and other analytical separation techniques, a plug of sample, also referred to as a sample pulse, is injected into the carrier flow at or near the channel inlet. Typically, a small volume of sample is injected to avoid dispersion or band broadening of the sample plug. Band broadening is detrimental as it reduces the resolution of separation. In current practice, the volume of the sample plug is limited by band broadening effects. The injected volume is typically 1-20 microliters, or less than 10% of the total volume of the FFF channel.
Field-flow fractionation is dissimilar to other analytical separation techniques because it utilizes an applied field for separation. Because of this feature, an additional sample introduction step is required for optimal resolution of separation. This process is the relaxation of the sample species with respect to the applied field. Equilibration is equivalently used in this context for relaxation. When the sample is first introduced into the FFF channel, it is generally distributed broadly over the channel cross section. Before the sample migration step is implemented, the sample species are subjected to a relaxation process in which they approach a steady-state distribution within the channel, usually by accumulating near one channel wall. The steady state distribution normally corresponds to a balance of the sample-field interaction which drives sample components towards the accumulation wall and Browrnan diffusion which drives sample away from the accumulation wall.
There are several methods for introducing sample into the field-flow fractionation channel. When referring to a sample, the terms "introducing", "injecting" or derivatives thereof are used as equivalent terms that encompass any procedure for incorporating into a carrier a sample that is to be separated or for introducing a flow into a conduit. Some methods provide a relaxed sample distribution. Other techniques merely position the sample components next to a wall without providing equilibration of the sample component with the field. The stop-flow method is the most commonly used method, and it provides a fully relaxed sample distribution. This method involves turning off the channel flow immediately following the sample injection and allowing the applied field to act upon the sample. This process both positions the sample at the wall and allows the sample components to equilibrate. The disadvantage of this method is that the carrier flow must be turned on and off; this typically requires a switching valve and extra time for equilibration. Furthermore, turning the flow on and off generates a pressure transient. The pressure transient generation is a most detrimental effect because the detectors used in FFF systems are sensitive to pressure transients. As a consequence of the pressure transient, the detector signal is distorted from its normal baseline value and a significant amount of time may be required for the detector to return to baseline. Whenever the detector response is disturbed, the separation cannot be accurately monitored, especially for species that elute at the beginning of the separation stage. Additionally, the pressure transient may broaden or otherwise disturb the sample zone which is precisely positioned in its equilibrium distribution during the previous stop-flow period. Either of these reasons will cause poor separation resolution. In addition to these undesired pressure pulses, a stop-flow process may also lead to another undesirable effect, which is adhesion of sample species at the accumulation wall.
A desirable feature of this method, however, is that the sample does not travel down the channel as it relaxes on the accumulation wall. This tends to reduce band broadening effects and broadening of the initial sample. Terms such as "dispersion", "broadening", "spreading", or equivalents thereof, will be used herein for describing the extension of the area or volume occupied by the sample whose components are to be separated. Focusing the sample is preventing sample spreading and thus avoiding the enlargement of the region occupied by the sample whose components are to be separated. The stop-flow method is described in Particle Size Distribution II, ACS Symposium Series No. 472, S. Kim Ratanathanawongs, Inho Lee, and J. Calvin Giddings, Separation and Characterization of 0.01-50-.mu.m Particles Using Flow Field-Flow Fractionation, 1991, chapter 15, pp. 229-46.
Some methods have been suggested for positioning the sample near the accumulation wall. U.S. Pat. No. 5,141,651 describes a pinched channel inlet system. In this method the thickness of the channel is reduced in the area of injection. Specifically, the structure of the channel is modified so that the position of the top or depletion channel wall is lowered. Consequently, the injected sample is, from the start, positioned closer to the accumulation wall. A pinched inlet channel system, however, has some shortcomings. First, since the flow through the channel is not discontinued in this method, the sample travels down the channel while also being relaxed towards the accumulation wall. This leads to increased band broadening effects and a broadened initial sample plug. Second, engineering the pinched inlet may present difficulties because high performance FFF channels are already very thin, typically 100-200 micrometers. Because of this small dimension, reducing the channel thickness near the inlet is difficult. Third, the reduced channel thickness in the pinched inlet must be even if the same flow velocity in all areas of the pinched inlet is to be maintained. Manufacturing a channel with an even channel thickness of just a few micrometers in the pinched inlet area is difficult. This dimension is determined by the typical thickness of an equilibrated sample zone, which is of the order of 1-10 micrometers. Fourth, at high channel flow rates, eddy currents may be generated at the interface between the pinched inlet area and the full channel thickness. Such eddy currents are undesirable because they may disturb the distribution of sample next to the accumulation wall. Finally, the reduced thickness of the channel at the inlet is susceptible to clogging.
Another process and apparatus for positioning sample near the accumulation wall are described in U.S. Pat. No. 5,193,688, and by Min-Kuang Liu, Stephen Williams, Marcus N. Myers, and J. Calvin Giddings, Hydrodynamic Relaxation in Flow Field-Flow Fractionation Using Both Split and Frit Inlets, Analytical Chemistry, Vol. 63 (1991), pp. 2115-22. This process is known as hydrodynamic sample relaxation, and it involves a permeable wall element, or frit inlet, positioned close to the sample inlet. This element is used to provide a separate flow stream that hydrodynamically forces sample to the accumulation wall. The permeable flow element is placed in the top channel wall, known as the depletion wall, and the flow from this element is distributed over the frit area immediately above the small inlet section of the channel where hydrodynamic relaxation is to be achieved. Flow is directed into this element using a separate pump and/or a flow control valve "tee-ed" into the carrier pump flow line. The amount of flow can be externally controlled to adjust the amount of viscous force that is applied to push the sample next to the accumulation wall. Thus, this relaxation process may be manipulated externally. In comparison to the pinched inlet, the channel structure required for hydrodynamic sample relaxation is easier to implement and is not subject to clogging. Nevertheless, this method has some disadvantages. First, a sample that relaxes according to this method is equilibrated relative to the field generated by the viscous force of flow through the permeable wall element. The magnitude of this hydrodynamically provided field is much larger than the field applied in the remainder of the channel. This increased magnitude is required by the necessity of positioning the sample at the accumulation wall very quickly to minimize band broadening effects. Because the hydrodynamic relaxation field typically does not match the field applied in the remainder of the channel, the sample must re-equilibrate when it is transported beyond the inlet region. Another disadvantage, common to the pinched channel inlet system, is that the sample has an increased opportunity for band broadening relative to the stop-flow method. This is because the carrier flow is not stopped during the relaxation process.
Hydrodynamic relaxation can also be pursued with a split inlet system. This system requires a splitter in the inlet region of the channel. The concepts underlying hydrodynamic relaxation, whether pursued with a frit inlet or with a split inlet system, are the same. A splitting plane is created in the region where two streams collide. The first stream is the carrier flow with the sample. The second stream is another flow that contains no sample, that is typically identical to the carrier flow, and that is introduced from above the carrier flow. The region where these two streams meet can be visualized as a plane, called the splitting plane. The flow rate of the second stream must exceed that of the sample stream for displacing the splitting plane--and with it all incoming particles--below the midplane of the channel. The greater the flow rate margin by which the second stream exceeds the stream that carries the sample, the closer the compression of the particles toward the accumulation wall, and the more complete the hydrodynamic relaxation. Equivalently, as this flow rate margin increases, the elevation of the splitting plane with respect to the accumulation wall decreases. The expected similarity in the results produced by the split inlet and frit inlet systems has been substantiated by Min-Kuang Liu, Stephen Williams, Marcus N. Myers, and J. Calvin Giddings, Hydrodynamic Relaxation in Flow Field-Flow Fractionation Using Both Split and Frit Inlets, Analytical Chemistry, Vol.63 (1991), pp. 2115 et seq.
The advantages common to both the pinched inlet and hydrodynamic relaxation techniques stem from the fact that the carrier flow need not be stopped. Thus, no pressure transient is generated, and the detector is not exposed to a pressure transient. In this context, the terms "detector" and "detector cell" are used interchangeably. The sample is also continually moving tangentially to the surface of the accumulation wall. This feature reduces the opportunity for sample adsorption on the accumulation wall. Furthermore, the pinched inlet and the hydrodynamic relaxation methods require a sufficiently short sample injection time to avoid sample diffusion during injection. Sample diffusion would otherwise form an undesirable and effectively larger sample plug.
Neither the pinched inlet nor the hydrodynamic relaxation method, however, provide complete sample relaxation. More specifically, neither one of these two methods controls the width of the sample plug, even though a compressed sample plug is desirable because it improves the resolution of the separation. A more compressed sample plug is provided by the stop flow method, which produces peaks that are sharper than those obtained with hydrodynamic relaxation. Equivalently, hydrodynamic relaxation results in somewhat broader elution bands than those produced by the stop flow technique. Furthermore, the sample in the stop flow technique is carried onto the channel by a carrier that occupies the full thickness of the channel, and the band does not undergo the spreading that is associated with the merging of the two streams in the frit inlet and split inlet techniques. Unfortunately, the stop flow method is more time consuming than stopless flow injection, it is more conducive to particle adhesion to the accumulation wall, and it typically produces a false signal due to the pressure pulses that are induced by abrupt flow changes in the channel.
For best results in field-flow fractionation, a minimum volume of sample should be introduced. Band broadening in hydrodynamic relaxation is increased by sample spreading as the sample flows into a split or a divided channel.
A method for providing a narrow initial sample plug is the outlet flow sample focusing method. This method has been practiced in tubular and rectangular cross section channels. The practice in tubular channels is described by H. L. Lee, J. F. G. Reis, J. Dohner, and E. N. Lightfoot, AIChE Journal, Vol. 20 (1974), pp. 776-84. The practice in rectangular cross section channels is described by K. G. Wahlund, and J. C. Giddings, Properties of an Asymmetrical Flow Field-Flow Fractionation Channel Having One Permeable Wall, Analytical Chemistry, Vol. 59 (1987), pp. 1332-39 and by S. K. R. Williams, and J. C. Giddings, Particle Size Analysis of Dilute Environmental Colloids by Flow Field-Flow Fractionation Using an Opposed Flow Sample Concentration Technique, Analytical Chemistry, Vol. 70 (1998), pp. 2495-2503. Whether tubular or rectangular cross section channels are used, the channels according to this method are constructed with one or more walls that are permeable to solvent flow. The tubular channel is a hollow fiber permeable to solvent flow. The rectangular channel used by Wahlund had a permeable bottom wall. The rectangular channel used by Williams had permeable top and bottom walls. According to the outlet flow sample focusing method, a flow additional and opposed to inlet flow is introduced from the outlet of the channel. Sample is introduced at or near the channel inlet, and the introduced sample is held stationary or is focused at the interface of the two opposing flows. The position of the interface is termed the sample focus plane and is related to the ratio of the two flow rates. Sample may be pumped in over a long period of time without causing a broad sample plug since the opposed flows continuously focus the sample. For tubular channels (for example, hollow fibers), sample is distributed radially to the outside perimeter of the tubular channel. For rectangular channels, sample is distributed towards the bottom channel wall. This process is capable of providing both a narrow and a fully equilibrated sample plug.
The disadvantage of the outlet flow sample focusing method lies in the transition that must be made between focusing and separation. During the focusing stage there is a forward directed flow from the channel and/or sample inlet(s) and backward directed flow coming in through the channel outlet. During the sample migration or separation stage, only forward directed channel inlet flow is implemented. This inlet flow carries the focused sample through the channel and out the outlet to a detector where the separated components are monitored. During the transition between focusing and separation, the outlet flow focusing procedure used by Lightfoot, Wahlund, and Williams requires that the direction of flow through the channel outlet be reversed. This requires a complex arrangement of pumps, tubing, and valves. At the transition between focusing and separation, which is the period during which the outlet flow is in the process of being reversed, a pressure transient is created in the channel. Additionally, the flow lines from the focusing point to the outlet of the channel must be established. During the transition between sample focusing and separation, the direction from the sample focus plane to the outlet must reverse completely. Thus, there is a period of unstable flow during the transition period. A consequence of this flow instability is that the focused sample is disturbed during the period of unstable flow; furthermore, the focused sample is also disturbed by the pressure transient. The detector is also disturbed by the pressure transient and the flow reversal. The effects on the detector may be slightly alleviated by placing a valve between the channel outlet and the detector so the backward directed focusing flow bypasses the detector cell. However, this requires extra valves and a pressure transient is generated due to the action of the valve.
Finally, an injector with minimal flow-interrupt transient is described in U.S. Pat. No. 4,506,558. This injector is a mechanical device that includes rotor and stator elements that can rotate relative to one another between load and injection positions. These elements have an interface and a series of conduits that run therethrough. The purpose of this mechanical device is to inject a sample at a high pressure into a chromatographic column, but it does not focus the injected sample.
Each of the afore-mentioned patents and references is hereby incorporated by reference in its entirety for the material disclosed therein.